Multifunctional carrier for the delivery of a pharmacological agent or genetic material into a cell

ABSTRACT

The present invention provides a drug delivery vehicle that can improve the pharmacokinetics of pharmacological agents. The invention relates to a multifunctional carrier capable of delivering a carried material such as a pharmacological agent or genetic material to a recipient. The multifunctional carrier includes a multifunctional core and a plurality of adduct molecules bonded thereto. The molecular carrier has surface functional groups which can be associated with a carried material. The carried material can be associated with the molecular carrier through covalent interactions or ionic interactions. The polyvalent core can be ethylene-diamine tetraacetic acid (EDTA) or succinic acid. The invention also relates to methods for producing and using such molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/310,492, which was filed on Aug. 8, 2001.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates generally to intracellular transports andtransport complexes and, in particular, to polyfunctional moleculescapable of delivering a desired pharmacological agent to a recipient.The invention also relates to methods for producing and using suchmolecules.

[0004] 2. Background of the Technology

[0005] Many valuable compounds must be discarded as potentialpharmaceuticals because of their pharmacokinetic profile. In some cases,the compound is cleared from circulation too rapidly, or accumulates toundesired levels in the kidneys or other organs, or is labile todigestive or circulatory enzymes or factors.

[0006] In light of the importance of surmounting the undesiredpharmacokinetics of many compounds, a variety of drug delivery systems,such as liposomes, microspheres, antibodies, etc., all aimed atimproving the therapeutic index and specificity of potentialpharmaceuticals have been suggested (U.S. Pat. No. 6,245,358 (Adami, etal.).

[0007] For example, various efforts to encapsulate or entrap the desiredpharmaceutical have been described (e.g., Yang, et al., “ControlledRelease Tacrine Delivery System For The Treatment Of Alzheimer'sDisease”, Drug Deliv., 8, 2, 93-98 (2001); Chandy, et al., “DevelopmentOf Poly (Lactic Acid)/Chitosan Co-Matrix Microspheres: ControlledRelease Of Taxol-Heparin For Preventing Restenosis”, Drug Deliv., 8, 2,77-86 (2001); Pignatello, et al., “Preparation And Analgesic Activity OfEudragit RS100 Microparticles Containing Diflunisal”, Drug Deliv., 8, 1,35-45 (2001). Likewise, liposome drug delivery formulations have beendescribed (Singh, et al., “Receptor-Mediated Gene Delivery To Hepg2Cells By Ternary Assemblies Containing Cationic Liposomes And CationizedAsialoorosomucoid”, Drug Deliv., 8, 1, 29-34 (2001); Petrikovics, etal., “In Vitro Studies On Sterically Stabilized Liposomes (SL) As EnzymeCarriers In Organophosphorus (OP) Antagonism”, Drug Deliv., 7, 2, 83-89(2001). Such approaches to drug delivery are reviewed by Gupta, et al.(“Biodegradable Polymer Microspheres As Vaccine Adjuvants And DeliverySystems”, Dev Biol Stand, 92, 63-78 (1998).

[0008] Additionally, active agents have been coupled or conjugated topolymeric compounds in an attempt to improve the delivery of the activeagent. For example, camptothecin has been conjugated to poly(L-glutamicacid) (PG) (Zou, et al., “Effectiveness Of Water Soluble Poly(L-GlutamicAcid)-Camptothecin Conjugate Against Resistant Human Lung CancerXenografted In Nude Mice”, Int J Oncol., 18, 2, 331-336 (2001). Theanticancer agent, paclitaxel, has likewise been conjugated topoly(L-glutamic acid) and found to possess improved pharmacokineticproperties (Oldham, et al., “Comparison Of Action Of Paclitaxel AndPoly(L-Glutamic Acid)-Paclitaxel Conjugate In Human Breast CancerCells”, Int J Oncol., 16, 1, 125-32 (2000). Similarly, Pechar, et al.describe the synthesis of a water-soluble polymer drug carrier systembased on biodegradable poly(ethylene glycol) block copolymer, and itsuse to produce a conjugate of doxorubicin. The copolymer consisted ofPEG blocks of molecular weight 2000 linked by means of an oligopeptidehaving amino end groups. Each of the oligopeptide blocks incorporated inthe carrier contained three carboxylic groups of which some were usedfor attachment of the doxorubicin via an enzymatically cleavabletetrapeptide spacer Gly-Phe-Leu-Gly (Pechar, et al., “Poly(EthyleneGlycol) Multiblock Copolymer As A Carrier Of Anti-Cancer DrugDoxorubicin”, Bioconjug Chem., 11, 2, 131-139 (2000). Drug polymerconjugates containing acid-cleavable bonds are reviewed by Kratz, etal., “Drug Polymer Conjugates Containing Acid-Cleavable Bonds”, Crit RevTher Drug Carrier Syst., 16, 3, 245-88 (1999). Such approaches to drugdelivery are reviewed by Sezaki, et al. (“Macromolecule-Drug ConjugatesIn Targeted Cancer Chemotherapy”, Crit Rev Ther Drug Carrier Syst., 1,1, 1-38 (1984).

[0009] Drug delivery systems comprising dendrimers and dendrimerconjugates are also known. See, for example, U.S. Pat. Nos. 4,289,872;4,360,646; 4,410,688, 4,587,329; 5,229,490; 5,338,532; 5,527,524;5,714,166; 5,882,645; and 6,194,543.

[0010] Despite such efforts, a need continues for a delivery vehiclethat can improve the pharmacokinetics of pharmacological agents. Thepresent invention is directed to such a need.

SUMMARY OF THE INVENTION

[0011] According to a first aspect of the invention, a molecular carriercomprising a central multivalent core and a plurality of adductmolecules bonded thereto is provided wherein the molecular carrier isrepresented by the formula:

[0012] wherein “Adduct” represents adduct moieties which can be the sameor different from one another and wherein the adduct moieties comprisethe residue of an amino acid. According to a second aspect of theinvention, the molecular carrier is represented by the formula:

[0013] or the formula:

[0014] wherein n is an integer of 2 or greater, m is 0 or a positiveinteger and p is a positive integer and wherein “Adduct” representsadduct moieties which can be the same or different from one another andwherein the adduct moieties comprise the residue of an amino acid.

[0015] According to a further aspect of the invention, one or more ofthe adduct moieties comprise a moiety represented by the formula:

[0016] or the formula:

[0017] wherein each R₁ and each R₂ independently represent a hydroxylgroup, a residue of an amino acid, or a polyfunctional amine. Accordingto a further aspect of the invention, one or more of the adduct moietiescomprise a moiety represented by the formula:

[0018] wherein each R₁ and each R₂ independently represent a hydroxylgroup, a residue of an amino acid, or a polyfunctional amine.

[0019] A pharmacological agent/molecular carrier complex is alsoprovided. The pharmacological agent/molecular carrier complex includes amolecular carrier as set forth above and a pharmacological agent whereinthe pharmacological agent is associated with one of the adduct moieties.The pharmacological agent can be a non-peptide drug, a protein, apeptide, a steroid or a hormone. For example, the pharmacological agentcan be a peptide antigen capable of eliciting an immune response, anopiate peptide an LHRH antagonist, a non-peptide drug such aspaclitaxel, or an angiogenic agent which can inhibit or stimulateangiogenesis.

[0020] The molecular carrier can include one or more maleimide groupsand the pharmacological agent can be covalently associated with themolecular carrier by reaction of a thiol group on the pharmacologicalagent with the maleimide group on the molecular carrier. Thepharmacological agent/molecular carrier complex can be dispersed in asolution as an emulsion or suspension or immobilized to a solid support.

[0021] A genetic material/molecular carrier complex comprising amolecular carrier as set forth above and genetic material, wherein thegenetic material is associated with one of the adduct moieties, is alsoprovided. The molecular carrier can have one or more positively chargedsurface functional groups and the genetic material can be ionicallyaccociated with the molecular carrier through the one or more positivelycharged surface functional groups. For example, the molecular carriercan include the residue of a polyfunctional amine and one or more aminegroups of the polyfunctional amine can be protonated to form thepositively charged surface functional groups.

[0022] A method of forming a molecular carrier is also provided. Themethod includes covalently bonding a plurality of adduct molecules to acentral multivalent core molecule by reacting a nucleophilic group oneach adduct molecule with an electrophilic group on the multivalent coremolecule. The multivalent core molecule comprises at least twoelectrophilic groups. According to a further embodiment of theinvention, the multivalent core molecule is a polyfunctional-carboxylicacid.

[0023] According to a further aspect of the invention, a molecularcarrier comprising a central multivalent core and a plurality of adductmolecules bonded thereto is provided wherein the central multivalentcore is the residue formed by the nucleophilic acyl substitutionreaction of each of the amino groups of a polyfunctional-amine with acarboxylic acid group of an adduct molecule. According to this aspect ofthe invention, the polyfunctional-amine can be: a benzene-tetramine;tri(carboxymethyl)amine; ((Lys)₂Lys)₃-(TFA), wherein TFA is atri-functional amine such as tris(2-aminoethyl)amine;diethylaminetriamine; triethylenetetramine; orNH₂((CH₂)_(n)NH)_(m)(CH₂)_(n)NH₂, wherein n and m are integers that maybe the same or different and which may vary throughout the molecule.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIGS. 1A and 1B depict schematic structures for preferredconjugates of the present invention.

[0025]FIGS. 2A through 2E illustrate preferred conjugates of the presentinvention wherein the core molecule of the multi-functional carrier isdenoted by a large circle, the adduct molecules are shown as smallercircles and the carried material (i.e., the pharmacological agent) isshown as an ellipsoid.

[0026]FIG. 3 schematically illustrates conjugates having cleavableadducts.

[0027]FIG. 4 shows a reaction scheme for producing [ETDA(Glu)₄].

[0028]FIG. 5 shows a reaction scheme for producing [Glu(Glu)₂].

[0029]FIG. 6 shows a reaction scheme for producing a conjugate having 16carboxylic acid groups.

[0030]FIG. 7 shows a synthesis of a Glu[Glu(Glu)₂]₂ product.

[0031]FIG. 8 shows conjugates of EDTA having 32 carboxylic acid groups.

[0032]FIG. 9 shows a synthesis of a Glu[Glu[Glu(Glu)₂]₂]₂ product.

[0033]FIG. 10 shows an EDTA adduct having 64 carboxylic acid groups.

[0034]FIG. 11 shows a synthesis of an EDTA-[Glu(Glu)₂]₄ multi-functionalcarrier.

[0035]FIG. 12 shows a synthesis of a Succinic Acid-[Glu[Glu(Glu)₂]₂]₂multi-functional carrier.

[0036]FIG. 13 shows a peptide covalently conjugated to amulti-functional carrier having an multivalent core.

[0037]FIG. 14 is a bar chart showing optical density at 405 nm for HPLCfractions of succinic acid-(Glu)_(n) adducts conjugated to peptide Tanalyzed by an ELISA.

[0038]FIG. 15 is a bar chart showing optical density at 405 nm for mousepolyclonal antibodies to succinic acid-(Glu)_(n) adducts conjugated topeptide T analyzed by an ELISA.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention provides a drug delivery vehicle that canimprove the pharmacokinetics of pharmacological agent. The inventionrelates to multifunctional molecules capable of deliveringpharmacological agents to a recipient. The invention also relates tomethods for producing and using such molecules.

[0040] In detail, the invention provides a drug delivery systemcomprising a molecular carrier, the carrier comprising a centralpolyvalent core, the core having a plurality of adduct molecules boundthereto, wherein each of the bound adduct molecules possesses multiplereactive groups for binding a desired pharmacological agent.

[0041] The invention further provides the embodiment of such drugdelivery system wherein the core molecule will array the bound adductmolecules in a symmetrical manner.

[0042] The invention further provides the embodiment of such drugdelivery system wherein the polyvalent core is bivalent, trivalent,tetravalent, pentavalent or hexavalent. The invention further providesthe embodiment of such drug delivery system wherein the polyvalent coreis bivalent, especially wherein the bivalent core is ethylenediamine.The invention further provides the embodiment of such drug deliverysystem wherein the polyvalent core is trivalent especially wherein thetrivalent core is tris (2-aminoethylamine), 1,2,3-propanetricarboxylicacid, 2-hydroxy propane-1,2,3-tricarboxylate, 1-hydroxypropane-1,2,3-tricarboxylate, tri(carboxymethyl)amine,diethylaminetriamine, ((Lys)₂Lys)₃-(TFA), ((Orni)₂ Orni)₃-(TFA),((DAB)₂DAB)₃-(TFA), or any combination of DAB (diaminobutyric acid),Orni (ornithene) or Lys (lysine) adducts covalently linked to atri-functional amine (TFA) such as tris (2-aminoethyl)amine. Theinvention further provides the embodiment of such drug delivery systemwherein the polyvalent core is tetravalent, especially wherein thetetravalent core is benzenetetracarboxylic acid, a benzenetetramine, acyclopentanetetracarboxylic acid, a butanetetracarboxylic acid, anethylenediamine tetraalkylcarboxylic acid, an ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N′,N′ tetraacetic acid, a1,2-Diaminocyclohexane-N,N,N′,N′ tetraacetic acid,((COOH)CH₂)₂N(CH₂)₃NCH₂COOH(CH₂)₃NH₂, (COOH)CH₂N(CH₂)₃NCH₂COOH(CH₂)₂NH₂,or triethylenetetramine. The invention further provides the embodimentof such drug delivery system wherein the tetravalent core isethylenediamine tetraacetate. The invention further provides theembodiment of such drug delivery system wherein the polyvalent core ispentavalent, especially wherein the pentavalent core isdiethylenetriamine-pentaacetic acid. The invention further provides theembodiment of such drug delivery system wherein the polyvalent core ishexavalent, especially wherein the hexavalent core is atriethylenetetraminehexaacetic acid. The invention further provides theembodiment of such drug delivery system wherein the polyvalent core isNH₂((CH₂)_(n)NH)_(m)(CH₂)_(n)NH₂, where n and m are integers that may bethe same or different, and which may vary throughout the molecule.

[0043] The invention further provides the embodiment of such drugdelivery system wherein the reactive groups of the adduct molecules areselected from the group consisting of amino groups and carboxylic acidgroups. The invention further provides the embodiment of such drugdelivery system wherein the adduct molecules are bivalent. The inventionfurther provides the embodiment of such drug delivery system wherein theamino or carboxylic acid groups are the amino or carboxylic acid groupsof an amino acid. The invention further provides the embodiment of suchdrug delivery system wherein the amino acid is glutamic acid, asparticacid, lysine or arginine.

[0044] The invention further provides the embodiment of such drugdelivery system wherein at least some of the adduct molecules are boundto other adduct molecules. The invention further provides the embodimentof such drug delivery system wherein at least some of the adductmolecules are associated with a pharmacological agent. The inventionfurther provides the embodiment of such drug delivery system wherein atleast some of the adduct molecules are associated with onepharmacological agent, and at least some of the adduct molecules areassociated with a different pharmacological agent. The invention furtherprovides the embodiment of such drug delivery system wherein thepharmacological agent is selected from the group consisting of smallorganic molecules, proteins, peptides, steroids and hormones.

[0045] According to a preferred embodiment of the invention, thepharmacological agent is a peptide antigen which is capable of elicitingan immune response. It is known that synthetic peptides can induceantibodies reactive with their cognate sequences in the native proteins(i.e., that synthetic peptide and native protein comprise the sameepitope). Specific antibodies are useful as reagents in variousinvestigations. Furthermore, peptide antigens made by known peptidesynthesis techniques are useful for producing immunogens and forimmunoprophylaxis and in affinity purification of proteins, antibodies,or other molecules. Peptide antigens are disclosed in U.S. Pat. No.5,882,645, which is hereby incorporated by reference in its entirety.

[0046] According to a further embodiment, a plurality of peptideantigens are conjugated to a molecular carrier according to theinvention. The peptide antigens can be covalently conjugated to themolecular carrier via reactive groups (e.g., amine or carboxylic acidgroups) on the peptide. For example, an amino group on the peptide canbe reacted with a carboxylic acid group on the molecular carrier or viceversa.

[0047] According to a further embodiment of the invention, thepharmacological agent is a pain killer such as an opiate peptide. Opiatepeptides can also be covalently conjugated to the molecular carrier viareactive groups (e.g., amine or carboxylic acid groups) on the peptide.

[0048] According to a further embodiment of the invention, thepharmacological agent is a contraceptive such as an LHRH antagonist.Various LHRH antagonists are known. For example, the LHRH antagonistcetrorelix is disclosed in Kovacs et al., “Comparison of Mechanisms ofAction of Leuteinizing Hormone-releasing Hormone (LHRH) AntagonistCetrorelix and LHRH Agonist Triptorelin on the Gene Expression ofPituitary LHRH Receptors in Rats”, PNAS, Vol. 98, No. 21 (2001).

[0049] According to a further embodiment of the invention, thepharmacological agent can be an angiogenic agent capable of modulatingthe development of blood vessels in mammals. Angiogenic agents aredisclosed in U.S. Pat. No. 6,121,236, which is hereby incorporated byreference in its entirety.

[0050] Other pharmacological agents which can be conjugated to molecularcarriers according to the invention include non-peptide drugs such aspaclitaxel. Paclitaxel, which is a known anti-cancer agent, can beconjugated to the molecular carrier using known techniques. Conjugatesof paclitaxel and glutamic acid are, for example, disclosed by Oldham,et al. in Int. J. Oncol., 16, 1, 125-32 (2000).

[0051] Additionally, a genetic material can be conjugated to themolecular carrier according to the invention. The transfer of geneticmaterial into cells has many potential uses as therapeutic and/ordiagnostic agents for human illness. Genetic material can be transfectedand subsequently transcribed and expressed to make new proteins withincells, replacing either aberrant proteins or absent proteins caused bygenetic errors. In addition, smaller pieces of genetic materialincluding either DNA or RNA, can be transfected into cells to alter cellfunction or edit specific messenger RNA's to correct other types ofgenetic defects. Further, synthetic genetic material, such as modifiedforms of antisense oligonucleotides, can be transferred into cells toblock the production of specific proteins. This may be useful insuppressing cells that grow abnormally, such as cancer cells, or in thealteration of normal cell functions, such as immunosuppression for organtransplantation. Small pieces of genetic material, such as aptimers, canalso function as drugs, and the transfer of these forms of nucleic acidscan specifically alter cellular functions in a manner similar to apharmaceutical.

[0052] The molecular carriers of the present invention may be complexedwith genetic material and used for gene therapy in mammals includinghumans. A method for preventing or treating a disease may comprisetransfecting a mammalian cell with a molecular carrier according to theinvention complexed with genetic material. As discussed earlier, geneticmaterial may be transfected into cells for a variety of reasonsincluding the production of proteins within cells, altering cellfunction, correcting genetic defects, and the like. In this manner,genetic diseases or conditions may be prevented or treated using thecomplex of a molecular carrier and genetic material according to thepresent invention.

[0053] When the carried material is genetic material, the surface of thedendrimer preferably comprises of a predominance of positively chargedsurface functional groups. The positive functionality can achieved byproviding amino surface functional groups on the molecular carrier. Whenthe surface functional groups of the molecular carrier are carboxylicacid groups, the positively charged functional groups can be introducedchemically. For example, polyfunctional amines such as ethylene diamine,spermine or spermidine can be reacted with the carboxylic acid surfacefunctional groups on the molecular carrier. Additionally, compoundscomprising both a nucleophilic group (e.g. an amine) and one or morepositively charged groups (e.g., quaternary onium salts) can be reactedwith the electrophilic carboxylic acid groups to provide the positivelycharged surface groups. The above materials are only exemplary and othercompounds and techniques can be used according to the invention toprovide positively charged surface functional groups on the molecularcarrier.

[0054] As set forth above, the carried material can be associatedcovalently or ionically with the multi-functional carrier. When thecarried material is a peptide having a cysteine residue, the peptide canbe associated with maleimide functional groups on the molecular carrier.These maleimide groups can be provided by reacting surface functionalamino groups on the carrier with compounds containing both maleimidegroups and electrophilic groups (e.g., carboxylic acid groups). Examplesof such compounds include 3-maleimidopropionic acid and4-(N-maleimidomethyl)cyclohexane-1-carboxylate. If the multifunctionalcarrier comprises nucleophilic (e.g., carboxylic acid) surfacefunctional groups, the amino groups can be provided by reacting thecarboxylic acid surface functional groups with a polyfunctional aminesuch as spermine, spermidine or ethylene-diamine.

[0055] The present invention further provides a drug delivery systemwherein the molecular carrier is soluble or solubilized. The inventionfurther provides the embodiment of such drug delivery system wherein themolecular carrier is immobilized to a solid support.

[0056] The invention further provides a method for improving thepharmacokinetic profile of a pharmacological agent which comprisesforming a delivery system comprising a molecular carrier, the carriercomprising a central polyvalent core, the core having a plurality ofadduct molecules bound thereto, wherein the wherein adduct moleculeshave reactive groups, and the pharmacological agent is bound to thereactive groups of the adduct molecules.

[0057] The present invention relates to a drug delivery systemcomprising a molecular carrier, which is composed of a centralpolyvalent core having a plurality of adduct molecules bound thereto,wherein each of the bound adduct molecules possesses multiple reactivegroups for binding a desired pharmacological agent.

[0058] The reactive groups of the adduct molecules are selected so thatat least one reactive group will be capable of binding (e.g., ionicallyor covalently) to a group of a desired pharmacological agent. Thepresence of the adduct molecules thus permits multiple molecules of thepharmacological agent to become associated to a single core.

[0059] As used herein, the term pharmacological agent is intended to bebroadly construed, and to encompass small organic molecules (e.g.,inhibitors, opiates, anti-inflammatory agents), as well as proteins(e.g., enzymes, clotting factors such as tissue plasminogen activator,etc.), peptides (e.g., protein mimetics, inhibitors, vaccines, etc.),steroids, hormones (e.g., insulin, progesterone, estrogen, etc.),nucleic acid molecules (DNA, and RNA), etc.

[0060] The term “conjugate molecules” as used herein is intended torefer to a core molecule associated with at least one, and preferably,two or more adduct molecules. The core molecule will be polyvalent, thushaving the ability to become associated with two or more, and preferablyfour or more adduct molecules. In a preferred embodiment, the coremolecule will array such adduct molecules in a symmetrical manner so asto minimize any steric interference, and maximize the solubility of thepharmacological agent-adduct-core complex. Examples of suitable coremolecules include bivalent, trivalent, tetravalent, pentavalent,hexavalent, etc. molecules including:

[0061] Bivalent Molecules:

[0062] ethylenediamine;

[0063] succinic acid;

[0064] diaminopropane; and

[0065] diaminobutane;

[0066] Trivalent Molecules:

[0067] Tris(2-aminoethyl)amine;

[0068] Tris(3-aminopropyl)amine;

[0069] Bis(3-aminopropyl)amine;

[0070] Spermidine;

[0071] Diethylenetriaminepentaacetic acid;

[0072] 1,2,3-Propanetricarboxylic acid;

[0073] 2-hydroxy-1-Hydroxypropane-1,2,3-tricarboxylate

[0074] Tri(carboxymethyl)amine;

[0075] Diethylaminetriamine;

[0076] Tetravalent Molecules:

[0077] Benzenetetracarboxylic acids, including:

[0078] 1,2,4,5 benzenetetracarboxylic acid

[0079] 1,2,3,5 benzenetetracarboxylic acid

[0080] 1,2,3,4 benzenetetracarboxylic acid

[0081] Benzenetetramines, including,

[0082] 1,2,4,5 benzenetetramine

[0083] Cyclopentanetetracarboxylic acid, including:

[0084] cis, cis, cis, cis, 1,2,3,4 cyclopentanetetracarboxylic acid

[0085] Butanetetracarboxylic acids, including:

[0086] 1,2,3,4 butanetetracarboxylic acid

[0087] Ethylenediamine tetraalkylcarboxylic acids, including:

[0088] ethylenediamine tetraacetate

[0089] Ethylene glycol-bis(beta-aminoethylether)-N,N,N′,N′ tetraaceticacid

[0090] 1,2-Diaminocyclohexane-N,N,N′,N′ tetraacetic acid, including:

[0091] trans-2-Diaminocyclohexane-N,N,N′,N′ tetraacetic acid

[0092] ((COOH)CH₂)₂N(CH₂)₃NCH₂COOH(CH₂)₃NH₂

[0093] (COOH)CH₂N(CH₂)₃NCH₂COOH(CH₂)₂NH₂

[0094] Triethylenetetramine; and

[0095] Spermine;

[0096] Pentavalent Molecules:

[0097] Diethylenetriaminepentaacetic acid; and

[0098] Spermidinepentaacetic acid;

[0099] Hexavalent Molecules:

[0100] Triethylenetetraminehexaacetic acid; and

[0101] Sperminehexaacetic acid;

[0102] ((Lys)₂Lys)₃-(TFA);

[0103] [(Ornithene)₂ornithene]₃-(TFA); and

[0104] [(Diamino butyric acid)₂ Diamino butyric acid]₃-(TFA).

[0105] “TFA” in the above formulae represents a tri-functional aminesuch as tris(2-aminoethyl)amine. Other polyvalent molecules that may beemployed comprise NH₂((CH₂)_(n)NH)_(m)(CH₂)_(n)NH₂, where n and m areintegers that may be the same or different, and which may varythroughout the molecule. Ethylenediamine tetraacetate (EDTA), succinicacid and ((Lys)₂Lys)₃-(TFA) are preferred core molecules.

[0106] The adduct molecules that may be used in accordance with thepresent invention will possess reactive groups, such as amino groups,carboxylic acid groups, etc. Preferably, the adduct molecules will bemultivalent, and as such have at least one group capable of associatingwith the core and a second group that is capable of associating with anadduct molecule or with a pharmacological agent. In a preferredembodiment, the reactive groups will be carboxylic acid groups. In apreferred embodiment, the adduct will be glutamic acid (Glu) or asparticacid (Asp), although other amino acids such as lysine (Lys) or arginine(Arg) may be employed. FIGS. 1A and 1B show molecular carrierscomprising an EDTA core and glutamic acid adducts (FIG. 1A) and an EDTAcore and glutamic and aspartic acid adducts (FIG. 1B).

[0107] Thus, the adducts conjugated to the core may be the same aminoacid as shown in FIG. 1A or, alternatively, mixtures of different aminoacids may be employed as shown in FIG. 1B. In a further embodiment, suchcarboxylic acid adducts can themselves be adducted by additional aminoacids, so as to produce peptide or pseudo-peptide carboxylic acidadducts, which each may be homogeneous or heterogeneous in composition,length and/or branching. In a preferred embodiment, the core willcomprise multiple adduct “strands” which may be branched or unbranched,and which may be connected or not connected to other strands attached tothe same core.

[0108] According to a pereferred embodiment of the invention, themolecular carrier is represented by the formula:

[0109] wherein “Adduct” represents adduct moieties which can be the sameor different from one another and wherein the adduct moieties comprisethe residue of an amino acid.

[0110] According to a further preferred embodiment of the invention, themolecular carrier is represented by the formula:

[0111] or by the formula:

[0112] wherein n is an integer of 2 or greater, m is 0 or a positiveinteger and p is a positive integer and wherein “Adduct” representsadduct moieties which can be the same or different from one another andwherein the adduct moieties comprise the residue of an amino acid.

[0113]FIGS. 2A through 2E depict schematic structures of exemplaryconjugates of the present invention illustrating this aspect of theinvention. As is apparent from the figures, all of the adduct moleculesneed not be associated with a pharmacological agent.

[0114] Any of a variety of reagents and methods can be used to bind theadduct molecules to the reactive groups of the core molecule. Mostpreferably, however, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) is employed for this purpose. EDC is a water-soluble derivative ofcarbodiimide. Carbodiimide catalyzes the formation of amide bondsbetween carboxylic acids or phosphates and amines by activating carboxylor phosphate to form an O-urea derivative. This derivative reactsreadily with nucleophiles. The reagent can be used to make ether linksfrom alcohol groups and ester links from acid and alcohols or phenols,and peptide bonds from acid and amines (Tedder, et al., “Amino-Acids andProteins”, In: Basic Organic Chemistry, Chapter 6, pp. 305-342, London,John Wiley & Sons (1972)). Carbodiimide is often used in the synthesisof peptides as the water-soluble derivative EDC or as the organicsoluble derivative, N,N′-dicyclohexyl-carbodiimide (DCC).

[0115] Additionally, the conjugates of the present invention may be usedto conjugate a single pharmacological agent, or two or more differentpharmacological agents. One aspect of the present invention concerns ameans for concentrating the delivery of a pharmacological agent to asite of need. In this regard, the conjugates of the present inventionmay have a pharmacological agent associated with one or more of itsadduct molecules and a receptor, receptor ligand, etc. associated withanother of such adduct molecules.

[0116] In one embodiment of the present invention, the associatedpharmacological agents of the conjugates of the present invention arereleased from the adducts by enzymes, and/or other factors presentwithin the tissues and organs of recipients, or by solubility ordecomposition. Thus, the conjugates of the present invention may be usedto effect the sustained release of the associated pharmacological agent,and thereby improve the pharmacokinetic profile of such agent.Preferably, such decomposition will be accomplished through the use ofadduct molecules that are labile to enzymes (for example, possessing acleavable disulfide bond as shown in FIGS. 3A and 3B. By using adductmolecules that are labile to intracellular enzymes, the conjugates ofthe present invention can be caused to decompose within cells. By usingadduct molecules that are labile to digestive enzymes, the conjugates ofthe present invention can be caused to decompose within the digestivetract, or within or near organs of the digestive system (e.g., stomach,colon, small intestine, gall bladder, etc.). By using adduct moleculesthat are labile to serum enzymes, the conjugates of the presentinvention can be caused to decompose within the circulatory system.

[0117] In an alternative embodiment, of the present invention, theassociated pharmacological agents of the conjugates of the presentinvention are not designed to be released from the conjugate. Thus, theconjugates of the present invention may be used to effect thelocalization or concentrating of the associated pharmacological agent atdesired sites within the recipient, and thereby improve thepharmacokinetic performance of such agent. For example, the conjugatemay be used with peptide or nucleic acid pharmacological agents toprovide improved vaccination. A conjugate designed to attack cancercells may, for example, comprise a tumor specific receptor bindingprotein and a pharmacological agent (such as an antitumor drug).

[0118] In one embodiment, the conjugates of the present invention willbe soluble or solubilized (i.e., formulated to be in an emulsion,suspension etc.). Such formulations will be capable of administering theconjugates to recipient patients buy oral, nasal, or inhalation routes,or intramuscularly, subcutaneously, transdermally, intravenously,intraurally or intraocularly. The conjugates of the present inventionmay similarly be used to provide pharmacological agents to cattle, pigs,sheep, dogs, cats, and other animals.

[0119] In an alternative embodiment, conjugates of the present inventioncontaining pharmacological agents may be immobilized onto solid supports(e.g., bandages, sutures, implants, laboratory and food servicesurfaces, etc.) so as to permit the sustained delivery of thepharmacological agent.

[0120] Having now generally described the invention, the same will bemore readily understood through reference to the following examples,which are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

Example 1 Synthesis of EDTA-Glutamic Acid Adducts

[0121] The principles of the present invention are illustrated withrespect to the synthesis of a preferred tetravalent conjugate molecule,which is formed through the coupling of EDC to EDTA.

[0122] EDTA Coupling (first shell)

[0123] (I) Ester synthesis:

[0124] To a 50 ml round-bottomed flask, 100 mg of EDTA tetrasodium saltand water (10 ml) was added. Glutamic acid diethyl ester (GDE)hydrochloride (264 mg) and 1-ether-3-(3-dimethylaminopropyl)carbodiimide(EDC) hydrochloride (211 mg) were then added. A clear solution wasformed. This solution was stirred at ambient temperature. Thirty minuteslater, the solution became cloudy. At this point tetrahydrofuran (THF)(10 ml) was added, and the solution turned clear again. Stirring wascontinued for 5 hours. The reaction mixture was then partitioned betweenethyl acetate (20 ml) and water (20 ml). The separated organic layer waswashed with brine (15 ml), dried over anhydrous sodium sulfate (5 g),and evaporated in a rotary evaporator under reduced pressure to give anoil, which was purified over silica gel flash chromatography (1:9methanol/ethyl acetate) to give the coupling product [EDTA(Glu)₄] as aviscous oil (Yield: 92%).

[0125] (II) Carboxylic Acid Synthesis (second shell):

[0126] The purified coupling product [EDTA(Glu)₄] (100 mg) was dissolvedin 5 ml THF, then 0.5 N aqueous sodium hydroxide (5 ml) was added. Theresulting mixture was stirred at ambient temperature for 2 hours. The pHof the solution was then adjusted to about 8 by the slow addition of 1.0N aqueous hydrogen chloride. The resulting solution was diluted withwater (50 ml), and then subjected to lyophilization and dialysis(molecular weight cutoff 500) to furnish the free acid (with 8carboxylic groups) as a white solid (Yield: not optimized). Thisreaction scheme is depicted in FIG. 4.

[0127] Block Synthesis

[0128] To a 100 ml round-bottomed flask, the following were successivelyadded: N-tert-butoxycarbony-glutamic acid (746 mg), water (25 ml), andsodium hydroxide (240 mg). Glutamic acid diethyl ester (GDE)hydrochloride (1.44 g) and 1-ethyl-3-(3-dimethylaminopropyl) carboiimide(EDC) hydrochloride (1.15 g) were then added. A clear solution wasformed. This solution was stirred at ambient temperature. Ten minuteslater the solution became cloudy. At this point THF (25 ml) was added,and the solution turned clear again. Stirring was continued for 5 hours.The reaction mixture was then partitioned between ethyl acetate (75 ml)and water (75 ml). The separated organic layer was washed with brine (50ml), dried over anhydrous sodium sulfate (20 mg) and evaporated in arotary evaporator under reduced pressure to give a colorless gel, whichwas purified over silica gel flash chromatography (1:1 ethylacetate/hexane, then ethyl acetate) to furnish the product [Glu(Glu)₂]as a colorless solid (Yield: 86%). This reaction scheme is depicted inFIG. 5.

[0129] Likewise, the [Glu(Glu)₂] product can be reacted with ETDAproduce larger conjugate molecules, such as a conjugate having 16carboxylic acid groups This embodiment is illustrated in FIG. 6.

[0130] In a similar manner, an n-tert butoxy [Glu(Glu)₂] product can bereacted with EDC and GDE hydrochlorides (as described above) to produce[Glu[Glu(Glu)₂]₂] as shown in FIG. 7. Such molecules can be reacted withEDTA to produce conjugates of EDTA having 32 carboxylic acid groups asillustrated in FIG. 8. Alternatively, the n-tert butoxy [Glu(Glu)₂]product can be reacted with EDC and the [Glu(Glu)₂] product to produce aGlu[Glu[Glu(Glu)₂]₂]₂ as shown in FIG. 9, which can be conjugated toEDTA to produce conjugates of EDTA having 64 carboxylic acid groups. Amolecular carrier having a multivalent core and 64 surface functionalcarboxylic acid groups is illustrated in FIG. 10.

[0131]FIG. 11 is an overview showing the synthesis of a conjugate of apeptide and an EDTA-[Glu(Glu)₂]₄ multi-functional carrier. Although amulti-functional carrier having 16 surface functional carboxylic acidgroups is shown, the procedure can be continued to provide a highergeneration multi-functional carrier having 32, 64, 128, etc. carboxylicacid groups. As shown in FIG. 13, a peptide can be covalently associatedwith the multi-functional carrier by reaction of an amino group on thepeptide with the carboxylic acid surface functional group on thecarrier.

Example 2 Synthesis of Succinic Acid-Glutamic Acid Adducts

[0132] Succ-Glu 1st Shell:

[0133] (I) Ester synthesis: To a 50 ML round-bottomed flask was added100 mg of succinic acid disodium salt (0.62 mmole) and water (10 ml).Glutamic acid diethyl ester hydrochloride (149 mg, 1.0 equivalent) wasadded in, followed by the addition of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride (119mg, 1.0 equivalent). A clear solution formed. This solution was stirredat ambient temperature. 30 minutes later, solution became cloudy, atthis moment tetrahydrofuran (THF) (10 ml) was added in, solution turnedclear again. Stirring was continued for 5 hours. The reaction mixturewas then partitioned between ethyl acetate (20 ml) and water (20 ml).The separated organic layer was washed with 0.1N aqueous HCl solution(2×15 ml), and brine (15 ml), dried over anhydrous sodium sulfate (5 g)and evaporated in a rotary evaporator under reduced pressure to give thedesired ester as a viscous oil. Yield: 95%. The molecular structure wasconfirmed by NMR and Mass Spectra.

[0134] (II) Carboxylic acid synthesis: The above ester was dissolved in5 ml tetrahydrofuran (THF), then 0.25 N aqueous sodium hydroxide (10 ml)was added. The resulting homogeneous solution was stirred at ambienttemperature for 2 hours. THF was evaporated in a rotary evaporator underreduced pressure. The remaining aqueous solution underwentlyophilization to furnish the desired acid (sodium salt) as a solid.Yield: 99%. Molecule structure was confirmed by NMR and Mass Spectra.

[0135] Peptide Preparation

[0136] Peptide T is an 8 amino acid peptide (mw=857) which is derivedfrom the V2 region of HIV-1, inhibits replication of R5 and dual-tropic(R5/X4) HIV-1 strains in monocyte-derived macrophages (MDMs), microglia,and primary CD4(+)T cells. Peptide T(D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH₂), a fragment corresponding toresidues 185-192 of gp120, the coat protein of HIV, is endowed withseveral biological properties in vitro, notably inhibition of thebinding of both isolated gp120 and HIV-1 to the CD4 receptor, andchemotactic activity. See, for example, Picone, et al., “Peptide TRevisited: Conformational Mimicry of Epitopes of Anti-HIV Proteins”, J.Pept. Sci., 7, 4, 197-207 (2001). The peptide T was syntheticallyprepared using a commercial available peptide synthesizer and purifiedby reverse phase HPLC. The purified peptide was reconstituted indistilled water to a final concentration of 1 mg/ml and stored at −80°C.

[0137] Synthesis of 2nd, 3rd, and 4th Shell:

[0138] The same procedure as described above was used except that theglutamic acid diethyl ester hydrochloride and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride wereused in two fold excess, respectively.

[0139] Preparation of EDTA/or Succinic Acid-(Glu)_(n)-(Peptide T)_(2n)

[0140] EDTA—Glu 1^(st) Shell and Peptide T: Acid: 0.5 mg Peptide T 4.0mg EDC 0.95 mg

[0141] EDTA—Glu 2^(nd) Shell and Peptide T: Acid: 0.5 mg Peptide T 3.5mg EDC 0.83 mg

[0142] Succ-Glu 1st Shell and Peptide T: Acid: 0.5 mg Peptide T 4.3 mgEDC 1.0 mg

[0143] Succ-Glu 2nd Shell and Peptide T: Acid: 0.5 mg Peptide T 3.6 mgEDC 0.86 mg

[0144] Succ-Glu 3rd Shell and Peptide T: Acid: 0.5 mg Peptide T 3.4 mgEDC 0.80 mg

[0145] Synthesis of EDTA-(Glu)_(n) or Succinic Acid-(Glu) Conjugateswith Peptide T

[0146] 0.5 mg of each EDTA/or succinic acid-(Glu)_(n) was dissolved in 1ml of 0.1 M MES (2-N-morpholino ethanesulfonic acid), pH=4.75 at roomtemperature. After 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) was added, the mixture was adjusted to pH=5.0 with0.1 N HCl solution, and stirred at room temperature for 10 minutes. Tothis solution, peptide T (1 mg/ml, dissolved in deionized water) wasadded and the mixture placed on a rocker and rocked gently at roomtemperature for 4 hours. The resulting mixture was dialyzed against 4liters deionized water 3 times with MWCO=3500 to remove free EDC. Thedistilled water was changed every 2 hours.

[0147]FIG. 12 is an overview showing the synthesis of a conjugate of apeptide and a Succinic Acid-[Glu[Glu(Glu)₂]₂]₂ multi-functional carrier.Although a multi-functional carrier having 16 surface functionalcarboxylic acid groups is shown, the procedure can be continued toprovide a higher generation multi-functional carrier having 32, 64, 128,etc. carboxylic acid groups. As shown in FIG. 13, the peptide can becovalently associated with the multi-functional carrier by reaction ofan amino group on the peptide with a carboxylic acid surface functionalgroup on the carrier.

[0148] The resulting peptide T conjugate solution was further purifiedby C18-RP-HPLC. HPLC Method of Purification of Peptide-T Conjugates Timeflow rate A (%) B (%) curve Initial 5.0 0 100 * 12 5.0 20   80  6   12.55.0 0 100 11 16 0.1 0 100 11

[0149] Mobile phase A: 90% CH₃CN, 10% H₂O, 0.05% TFA

[0150] Mobile phase B: H₂O, 0.05% TFA

[0151] Phenomenex Jupitor 250×10 mm, C18, 5u, 300 A;

[0152] UV detector, detection wavelength: 225 nm,

[0153] Analysis of Conjugated Peptide T

[0154] HPLC fractions of succinic acid-(Glu)_(n) conjugated to peptide Twere analyzed by ELISA (i.e., enzyme linked immunosorbent assay). HPLCfractions of conjugated peptide T were diluted (1:10) in coating buffer[1.59 g sodium carbonate (Na2CO3), 2.93 g sodium bicarbonate (NaHCO3)Dissolve in 900 ml H2O, adjust pH to 9.6 with HCl and make up to 1 L].Control net peptide T was dissolved in coating buffer with aconcentration of 5 μg/ml. 100 μl of each of two peptide T preparationswas added to each well of a 96-well microtiter plate (Nunc). The platewas covered with a plate sealer and incubated at 4° C. over night.Following 3 washes with PBS-Tween [phosphate buffer saline: 8.0 g sodiumchloride (NaCl), 0.2 g monobasic potassium phosphate (KH₂PO₄), 1.15 gdibasic sodium phosphate (Na₂HPO₄), 0.2 g potassium chloride (KCl),Dissolve in 900 ml H₂O, adjust pH to 7.4 with NaOH or HCl and make up to1 L, PBS+0.5 ml Tween 20 per liter]. 100 μl of diluted rabbit antipeptide T antiserum (1:250, in assay diluent, 0.1% BSA and 0.1% sodiumazide (NaN₃) in PBS) was added to each well. The plate was sealed andincubated for 1 hour at room temperature. Following 5 washes, each wellwas added with 100 μl of diluted goat anti rabbit IgG alkalinephosphatase conjugate (KPL, 1:1000 dilution in assay diluent). The platewas sealed and incubated for 30 min. Following final washes, 100 μl ofsubstrate solution [p-nitrophenyl phosphate from Sigma, 20 mg dissolvedin 10 ml substrate buffer, 0.844 g sodium bicarbonate (NaHCO₃), 1.255 gsodium carbonate (Na₂CO₃), 0.203 g magnesium chloride (MgCl₂.6H₂O),dissolve in 1000 ml H₂O] was added to each well. Optical density (OD)was then read at 405 nm.

[0155] The results are shown in FIG. 14 which is a bar chart showingoptical density at 405 nm of different HPLC fractions (F1, F2 and F3)for various Succinic Acid-(Glu)_(n) adducts conjugated to Peptide T andfor a control. In FIG. 14, Shell 1 refers to SuccinicAcid-(Glu)₂-(Peptide T)₄ conjugates; Shell 2 refers to SuccinicAcid-(Glu)₂-(Glu)₄-(Peptide T)₈ conjugates; Shell 3 refers to SuccinicAcid-(Glu)₂-(Glu)₄-(Glu)₈-(Peptide T)₁₆ conjugates; F refers to the HPLCfraction with F1 being the fraction with the lowest retention time andF3 being the fraction with the highest retention time; and PT refers toFree peptide-T. The results shown in FIG. 14 indicate that there aredifferent populations of peptide T conjugated to each adduct. For theShell 2 and Shell 3 adducts, as the amount of peptide T coupled to themolecular carrier increases (which is shown by its immunoreactivity),the retention time of HPLC increases (i.e., from Fraction #1 to Fraction#3). That is, for the Shell 3 adduct, Fraction 3, which has the highestretention time, also has the highest immunoreactivity. Similarly, forthe Shell 2 adduct, Fraction 2, which has the highest retention time,also has the highest immunoreactivity. Measuring molecular weight ofeach fraction using Mass Spectrum analysis should reveal the actualcoupling efficiency of the peptide to the molecular carrier.

[0156] Immunization Procedures for Mouse Polyclonal Antibodies to theSuccinic Acid-(Glu)n-Peptide T Conjugate

[0157] Balb/c mice were immunized intraperitoneally with succinicacid-(glu)n-Peptide T at 0.5 mg per immunization and per animal. Theantigen complex was administered with complete Freund's adjuvant (firstinjection) and with incomplete Freund's adjuvant for booster injections.Mice were immunized 3 times at day 0, day 8 and day 21. Mice were bledvia tail vein and 15 μl of blood collected by a capillary tube wastransferred into 135 μl PBS in a 12 X 75 tissue culture tube. Dilutedmouse sera were collected after centrifugation for 10 min.

[0158] ELISA Procedures for Mouse Polyclonal Antibodies to the SuccinicAcid-(Glu)n-Peptide T Conjugate

[0159] A polystyrene 96-well plate (Nunc) was coated with free peptide Tat 10 μg/ml in coating (pH 9.6) overnight at 4° C. The plate was washed3 times with wash buffer. Afterwards, 100 μl of diluted (1:100 in assaydiluent) mouse antiserum from each mouse was then added to each well(each serum sample was assayed in duplicate) and incubated for 2 hoursat room temperature. The plates were then washed 5 times with washbuffer. Goat anti-mouse IgG conjugated to alkaline phosphate (KPL) wasdiluted 1:1000 in PBS with 1% bovine serum albumin, added to the wells,and incubated for 30 min at room temperature followed by six washes withwash buffer. Substrate solution (p-Nitrophenyl phosphate, 2 mg/ml insubstrate buffer) was then added. Absorbance was then measured using amicroplate reader at 405 nm. The results are shown in FIG. 15 whereineach bar represents an average OD value of one mouse antiserum.

[0160] The ELISA results shown in FIG. 15 demonstrate that peptide T,when conjugated to different succinic acid-(glu)_(n) shells, induced asignificant immune response in mice. Further, some mice show greaterimmune response to both Shell 2-Peptide T and Shell 3-Peptidestimulations than other animals.

[0161] All publications and patent applications mentioned in thespecification are herein incorporated by reference to the same extent asif each individual publication or patent application had beenspecifically and individually indicated to be incorporated by reference.The discussion of the background to the invention herein is included toexplain the context of the invention. Such explanation is not anadmission that any of the material referred to was published, known, orpart of the prior art or common general knowledge anywhere in the worldas of the priority date of any of the aspects listed above.

[0162] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and that this application is intended to coverany variations, uses, or adaptations of the invention following, ingeneral, the principles of the invention and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the invention pertains and as may be applied tothe essential features hereinbefore set forth.

What is claimed is:
 1. A molecular carrier comprising a centralmultivalent core and a plurality of adduct molecules bonded thereto,wherein the molecular carrier is represented by the formula:

wherein “Adduct” represents adduct moieties which can be the same ordifferent from one another and wherein the adduct moieties comprise theresidue of an amino acid.
 2. The molecular carrier of claim 1, whereinone or more of the adduct moieties comprise a moiety represented by theformula:

wherein each R₁ and each R₂ independently represent a hydroxyl group, aresidue of an amino acid or a polyfunctional amine.
 3. The molecularcarrier of claim 1, wherein one or more of the adduct moieties comprisea moiety represented by the formula:

wherein each R₁ and each R₂ independently represent a hydroxyl group, aresidue of an amino acid or a polyfunctional amine.
 4. The molecularcarrier of claim 1, wherein one or more of the adduct moieties comprisea moiety represented by the formula:

wherein each R₁ and each R₂ independently represent a hydroxyl group, aresidue of an amino acid or a polyfunctional amine.
 5. A pharmacologicalagent/molecular carrier complex comprising the molecular carrier ofclaim 1 and a pharmacological agent, wherein the pharmacological agentis associated with at least one of the adduct moieties.
 6. Thepharmacological agent/molecular carrier complex of claim 5, furthercomprising a second pharmacological agent associated with at least oneof the adduct moieties, wherein the second pharmacological agent isdifferent than the first pharmacological agent.
 7. The pharmacologicalagent/molecular carrier complex of claim 5, further comprising areceptor or a receptor ligand associated with at least one of the adductmoieties.
 8. The pharmacological agent/molecular carrier complex ofclaim 6, wherein the adduct moiety with which the pharmacological agentis associated comprises an enzyme labile bond.
 9. The pharmacologicalagent/molecular carrier complex of claim 8, wherein the enzyme labilebond is labile to intracellular enzymes, digestive enzymes or serumenzymes.
 10. The pharmacological agent/molecular carrier complex ofclaim 8, wherein the enzyme labile bond is a disulfide linkage.
 11. Thepharmacological agent/molecular carrier complex of claim 7, furthercomprising a receptor binding protein associated with one of the adductmoieties.
 12. The pharmacological agent/molecular carrier complex ofclaim 5, wherein the pharmacological agent is selected from the groupconsisting of non-peptide drugs, proteins, peptides, steroids andhormones.
 13. The pharmacological agent/molecular carrier complex ofclaim 5, wherein the pharmacological agent comprises a peptide antigencapable of eliciting an immune response.
 14. The pharmacologicalagent/molecular carrier complex of claim 5, wherein the pharmacologicalagent comprises an opiate peptide.
 15. The pharmacologicalagent/molecular carrier complex of claim 5, wherein the pharmacologicalagent comprises an LHRH antagonist.
 16. The pharmacologicalagent/molecular carrier complex of claim 5, wherein the pharmacologicalagent comprises paclitaxel.
 17. The pharmacological agent/molecularcarrier complex of claim 5, wherein the pharmacological agent comprisesan angiogenic agent which inhibits or stimulates angiogenesis.
 18. Thepharmacological agent/molecular carrier complex of claim 5, wherein themolecular carrier comprises one or more maleimide groups and thepharmacological agent comprises a thiol group, and wherein thepharmacological agent is covalently associated with the molecularcarrier by reaction of the thiol group on the pharmacological agent withthe maleimide group on the molecular carrier.
 19. The pharmacologicalagent/molecular carrier complex of claim 5, wherein the pharmacologicalagent/molecular carrier complex is dispersed in a solution as anemulsion or suspension.
 20. The pharmacological agent/molecular carriercomplex of claim 5, wherein the molecular carrier is immobilized to asolid support.
 21. A genetic material/molecular carrier complexcomprising the molecular carrier of claim 1 and genetic material,wherein the genetic material is associated with one of the adductmoieties.
 22. The genetic material/molecular carrier complex of claim21, wherein the molecular carrier comprises one or more positivelycharged surface functional groups and wherein the genetic material isionically accociated with the molecular carrier through the one or morepositively charged surface functional groups.
 23. The geneticmaterial/molecular carrier complex of claim 22, wherein the molecularcarrier comprises the residue of a polyfunctional amine and wherein oneor more amine groups of the polyfunctional amine are protonated to formthe one or more positively charged surface functional groups.
 24. Thegenetic material/molecular carrier complex of claim 22, wherein thegenetic material is selected from the group consisting of DNA, RNA,oligonucleotides, and nucleic acids.
 25. A molecular carrier comprisinga central multivalent core and a plurality of adduct molecules bondedthereto, wherein the molecular carrier is represented by the formula:

or by the formula

wherein n is an integer of 2 or greater, m is 0 or a positive integerand p is a positive integer and wherein “Adduct” represents adductmoieties which can be the same or different from one another and whereinthe adduct moieties comprise the residue of an amino acid.
 26. Themolecular carrier of claim 25 wherein the molecular carrier isrepresented by the formula:

wherein the adduct moieties comprise the residue of an amino acid. 27.The molecular carrier of claim 25, wherein one or more of the adductmoieties comprise a moiety represented by the formula:

wherein each R₁ and each R₂ independently represent a hydroxyl group, aresidue of an amino acid, or a polyfunctional amine.
 28. The molecularcarrier of claim 25, wherein one or more of the adduct moieties comprisea moiety represented by the formula:

wherein each R₁ and each R₂ independently represent a hydroxyl group, aresidue of an amino acid, or a polyfunctional amine.
 29. The molecularcarrier of claim 25, wherein one or more of the adduct moieties comprisea moiety represented by the formula:

wherein each R₁ and each R₂ independently represent a hydroxyl group, aresidue of an amino acid or a polyfuctional amine.
 30. A pharmacologicalagent/molecular carrier complex comprising the molecular carrier ofclaim 25 and a pharmacological agent, wherein the pharmacological agentis associated with at least one of the adduct moieties.
 31. Thepharmacological agent/molecular carrier complex of claim 30, furthercomprising a second pharmacological agent associated with at least oneof the adduct moieties.
 32. The pharmacological agent/molecular carriercomplex of claim 30, further comprising a receptor or a receptor ligandassociated with at least one of the adduct moieties.
 33. Thepharmacological agent/molecular carrier complex of claim 30, wherein theadduct moiety with which the pharmacological agent is associatedcomprises an enzyme labile bond.
 34. The pharmacological agent/molecularcarrier complex of claim 33, wherein the enzyme labile bond is labile tointracellular enzymes, digestive enzymes or serum enzymes.
 35. Thepharmacological agent/molecular carrier complex of claim 33, wherein theenzyme labile bond is a disulfide linkage.
 36. The pharmacologicalagent/molecular carrier complex of claim 32, further comprising areceptor binding protein associated with one of the adduct moieties. 37.The pharmacological agent/molecular carrier complex of claim 30, whereinthe pharmacological agent is selected from the group consisting ofnon-peptide drugs, proteins, peptides, steroids and hormones.
 38. Thepharmacological agent/molecular carrier complex of claim 30, wherein thepharmacological agent comprises a peptide antigen capable of elicitingan immune response.
 39. The pharmacological agent/molecular carriercomplex of claim 30, wherein the pharmacological agent comprises anopiate peptide.
 40. The pharmacological agent/molecular carrier complexof claim 30, wherein the pharmacological agent comprises an LHRHantagonist.
 41. The pharmacological agent/molecular carrier complex ofclaim 30, wherein the pharmacological agent comprises paclitaxel. 42.The pharmacological agent/molecular carrier complex of claim 30, whereinthe pharmacological agent comprises an angiogenic agent which inhibitsor stimulates angiogenesis.
 43. The pharmacological agent/molecularcarrier complex of claim 30, wherein the molecular carrier comprises oneor more maleimide groups and the pharmacological agent comprises a thiolgroup, and wherein the pharmacological agent is covalently associatedwith the molecular carrier by reaction of the thiol group on thepharmacological agent with the maleimide group on the molecular carrier.44. The pharmacological agent/molecular carrier complex of claim 30,wherein the pharmacological agent/molecular carrier complex is dispersedin a solution as an emulsion or suspension.
 45. The pharmacologicalagent/molecular carrier complex of claim 30, wherein the molecularcarrier is immobilized to a solid support.
 46. The molecular carrier ofclaim 25, wherein at least some of the adduct molecules are bonded toother adduct molecules.
 47. A genetic material/molecular carrier complexcomprising the molecular carrier of claim 25 and genetic material,wherein the genetic material is associated with one of the adductmoieties.
 48. The genetic material/molecular carrier complex of claim47, wherein the molecular carrier comprises one or more positivelycharged surface functional groups and wherein the genetic material isionically accociated with the molecular carrier through the one or morepositively charged surface functional groups.
 49. The geneticmaterial/molecular carrier complex of claim 48, wherein the molecularcarrier comprises the residue of a polyfunctional amine and wherein oneor more amine groups of the polyfunctional amine are protonated to formthe one or more positively charged surface functional groups.
 50. Thegenetic material/molecular carrier complex of claim 47, wherein thegenetic material is selected from the group consisting of DNA, RNA,oligonucleotides, and nucleic acids.
 51. A method of forming a molecularcarrier comprising: covalently bonding a plurality of adduct moleculesto a central multivalent core molecule by reacting a nucleophilic groupon each adduct molecule with an electrophilic group on the multivalentcore molecule, wherein the multivalent core molecule comprises at leasttwo electrophilic groups.
 52. The method of claim 51, wherein themultivalent core molecule is a polyfunctional-carboxylic acid.
 53. Themethod of claim 51, wherein the multivalent core is succinic acid orethylene diamine tetraacetic acid (EDTA).
 54. The method of claim 51,wherein the nucleophilic group on each adduct molecule is an aminogroup.
 55. The method of claim 51, wherein the adduct molecules compriseglutamic acid, aspartic acid or adducts thereof.
 56. The method of claim55, wherein the adduct molecules comprise the tetra-ester of Glu(Glu)₂,the octa-ester of [Glu[Glu(Glu)₂]₂], the hexadeca-ester of[Glu[Glu[Glu(Glu)₂]₂]₂] or esters of higher order adducts of glutamicacid.
 57. The method of claim 55, wherein the adduct molecules compriseglutamic acid ester or aspartic acid ester adducts, the method furthercomprising a step of hydrolysing the ester groups on the adductmolecules to the corresponding carboxylic acid groups.
 58. The method ofclaim 51, wherein one or more adduct molecules comprise enzyme labilelinkages.
 59. The method of claim 58, wherein the enzyme labile linkagescomprise disulfide linkages.
 60. The method of claim 55, furthercomprising: a) reacting a first N-substituted glutamic or aspartic acidwith 2 equivalents of a glutamic or aspartic acid di-ester andde-protecting the amine group on the first glutamic or aspartic acid toform a first glutamic or aspartic acid di-ester adduct; b) optionallyreacting a second N-substituted glutamic or aspartic acid with twoequivalents of the first glutamic or aspartic acid di-ester adduct andde-protecting the amine group on the second glutamic or aspartic acid toform a second glutamic or aspartic acid di-ester adduct; and c)optionally reacting a third N-substituted glutamic or aspartic acid withtwo equivalents of the second glutamic or aspartic acid di-ester adductand de-protecting the amine group on the third glutamic or aspartic acidto form a third glutamic or aspartic acid di-ester adduct; wherein theadduct molecules covalently bonded to the multivalent core independentlycomprise the first, second and/or third glutamic or aspartic aciddi-ester adducts.
 61. The method of claim 51, further comprising a stepof associating a pharmacological agent with the molecular carrier. 62.The method of claim 61, wherein the associating step comprisescovalently bonding the pharmacological agent to the molecular carrier.63. The method of claim 61, further comprising associating a receptor ora receptor ligand with the molecular carrier.
 64. The method of claim51, wherein the reacting step is conducted in the presence of acatalyst.
 65. The method of claim 64, wherein the catalyst is acarbodiimide.
 66. The method of claim 65, wherein the carbodiimide is1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) orN,N′-dicyclohexyl-carbodiimide (DCC).
 67. The method of claim 51,wherein the molecular carrier comprises carboxylic acid surfacefunctional groups, the method further comprising: reacting one or moreof the surface functional carboxylic acid groups on the molecularcarrier with a polyamine to provide one or more amine surface functionalgroups; and ionically complexing genetic material to the one or moreamine surface functional groups.
 68. The method of claim 51, wherein themolecular carrier comprises carboxylic acid surface functional groups,the method further comprising: reacting a compound comprising amaleimide group and a carboxylic acid reactive functional group with oneor more of the surface functional groups to provide one or moremaleimide surface functional group; and covalently attaching apharmacological agent to the molecular carrier by reacting a thiol groupon the pharmacological agent with the one or more maleimide groups onthe molecular carrier.
 69. The method of claim 68, wherein thepharmacological agent is a peptide containing a cysteine residue.
 70. Amolecular carrier made by the method of claim
 51. 71. A pharmacologicalagent/molecular carrier complex made by the method of claim
 61. 72. Agenetic material/molecular carrier complex made by the method of claim67.
 73. The molecular carrier of claim 70, wherein the core molecule isselected from the group consisting of: a benzene-tetracarboxylic acid; acyclopentane-tetracarboxylic acid; a butane-tetracarboxylic acid; anethylenediamine tetraalkylcarboxylic acid; an ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N′,N′ tetraacetic acid; a1,2-Diaminocyclohexane-N,N,N′,N′ tetraacetic acid;spermidine-pentaaceticacid; spermine-hexaaceticacid;((COOH)CH₂)₂N(CH₂)₃NCH₂COOH(CH₂)₃NH₂; and(COOH)CH₂N(CH₂)₃NCH₂COOH(CH₂)₂NH₂.
 74. A method of forming a molecularcarrier comprising: covalently bonding a plurality of adduct moleculesto a central multivalent core molecule by reacting an electrophilicgroup on each adduct molecule with a nucleophilic group on themultivalent core molecule, wherein the multivalent core moleculecomprises at least two nucleophilic groups, and wherein the multivalentcore molecule is selected from the group consisting of: abenzene-tetramine; a tri(carboxymethyl)amine; [(Lys)₂Lys]₃-(TFA),wherein TFA is a tri-functional amine; diethylaminetriamine;triethylenetetramine; Tris(hydroxymethyl)aminomethane (TRIS); andNH₂((CH₂)_(n)NH)_(m)(CH₂)_(n)NH₂, wherein n and m are integers that maybe the same or different and which may vary throughout the molecule. 75.The method of claim 74, wherein the adduct molecules comprise lysine orarginine residues, the method further comprising: a) reacting a firstlysine or arginine ester molecule with 2 equivalents of lysine orarginine and hydrolysing the ester group on the first lysine or arginineto a carboxylic acid to form a first lysine or arginine adduct; b)optionally reacting a second lysine or arginine ester with twoequivalents of the first lysine or arginine adduct and hydrolysing theester group on the second lysine or arginine ester to a carboxylic acidto form a second lysine or arginine adduct; and c) optionally reacting athird lysine or arginine ester with two equivalents of the second lysineor arginine adduct and hydrolysing the ester group on the third lysineor arginine ester to a carboxylic acid to form a third lysine orarginine adduct; wherein the adduct molecules covalently bonded to themultivalent core independently comprise the first, second and/or thirdlysine or arginine adducts.
 76. A molecular carrier made by the methodof claim
 74. 77. The molecular carrier of claim 76, wherein the adductmolecules comprise lysine or arginine residues.
 78. A pharmacologicalagent/molecular carrier complex comprising the molecular carrier ofclaim 73 and a pharmacological agent, wherein the pharmacological agentis associated with the molecular carrier.
 79. A pharmacologicalagent/molecular carrier complex comprising the molecular carrier ofclaim 76 and a pharmacological agent, wherein the pharmacological agentis associated with the molecular carrier.
 80. A geneticmaterial/molecular carrier complex comprising the molecular carrier ofclaim 76 and genetic material, wherein the genetic material isassociated with the molecular carrier.
 81. The geneticmaterial/molecular carrier complex of claim 80, wherein the geneticmaterial is ionically complexed to one or more amine surface functionalgroups of the molecular carrier.
 82. A method for introducing asubstance into cells comprising incubating one or more cells with amolecular carrier as set forth in claim 1 associated with apharmacological agent or a genetic material.
 83. The method of claim 82,wherein the molecular carrier is associated with a pharmacological agentselected from the group consisting of non-peptide drugs, proteins,peptides, steroids and hormones.
 84. The method of claim 82, wherein themolecular carrier is associated with a genetic material selected fromthe group consisting of DNA, RNA, oligonucleotides, and nucleic acids.85. A method for introducing a substance into cells comprisingincubating one or more cells with a molecular carrier as set forth inclaim 25 associated with a pharmacological agent or a genetic material.86. The method of claim 85, wherein the molecular carrier is associatedwith a pharmacological agent selected from the group consisting ofnon-peptide drugs, proteins, peptides, steroids and hormones.
 87. Themethod of claim 85, wherein the molecular carrier is associated with agenetic material selected from the group consisting of DNA, RNA,oligonucleotides, and nucleic acids.
 88. A method for in vivotransfection of cells comprising administering to the cells a molecularcarrier as set forth in claim 1 associated with a pharmacological agentor a genetic material.
 89. A method for in vivo transfection of cellscomprising administering to the cells a molecular carrier as set forthin claim 25 associated with a pharmacological agent or a geneticmaterial.
 90. A method for performing gene therapy comprisingadministering a gene therapy agent to a human or animal subject, whereinthe gene therapy agent comprises a molecular carrier as set forth inclaim 1 associated with a genetic material.
 91. The method of claim 90,wherein the genetic material comprises an expression vector containing aDNA segment encoding a protein or an anti-sense oligonucleotide.
 92. Amethod for performing gene therapy comprising administering a genetherapy agent to a human or animal subject, wherein the gene therapyagent comprises a molecular carrier as set forth in claim 25 associatedwith a genetic material.
 93. The method of claim 92, wherein the geneticmaterial comprises an expression vector containing a DNA segmentencoding a protein or an anti-sense oligonucleotide.